Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and vasp phosphatases

Abstract

The invention relates to an HTS-appropriate test system for detecting the activity of cyclo-nucleotide-dependent protein kinases (cNPK) containing: a) at least one test compound; b) at least one appropriate cNPK substrate; c) at least one composition, which is to be incubated and which contains cNPK and ATP, optionally, phosphorylation reaction stoppers, and; d) an appropriate detection system for quantifying the phosphorylation of the cNPK substrate. The invention also relates to an HTS-appropriate test system for detecting the activity of VASP phosphatases containing: e) at least one test compound; f) at least one appropriate VASP phosphatase substrate; g) at least one composition, which is to be incubated and which contains VASP phosphatase, and; h) an appropriate detection system for quantifying the dephosphorylation of the VASP phosphatase substrate. The described test systems can be used for locating compounds, which modulate the activity of a cNPK or of a VASP phosphatase, from chemical or natural substance libraries.

1 . An HTS-suitable test system for detecting the activity of the cyclonucleotide-dependent protein kinases (cNPKs), comprising 
 a) at least one test compound,    b) at least one suitable cNPK substrate (cNPKS),    c) at least one composition which is to be incubated and which contains cNPK and ATP, and    d) a suitable detection system for quantitatively determining the quantity of the phosphorylated cNPK substrate.    
     
     
         2 . An HTS-suitable test system for detecting the activity of the VASP phosphatase, comprising 
 e) at least one test compound,    f) at least one suitable VASP phosphatase substrate,    g) at least one composition which is to be incubated and which contains VASP phosphatase, and    h) a suitable detection system for quantitatively determining the quantity of the dephosphorylated VASP phosphatase substrate.    
     
     
         3 . A test system according to  claim 1  or  2 , which can be automated and is heterogeneous, with separation or immobilization of at least one of the reaction products or detection reagents.  
     
     
         4 . A test system according to  claim 1  or  2 , which can be automated and is homogeneous.  
     
     
         5 . A test system according to any of  claims 1  to  4 , which is nonradioactive.  
     
     
         6 . A test system according to  claim 1  or  2 , characterized in that the cNPK substrate according to feature (b) or the VASP phosphatase substrate according to feature (f) is selected from SEQ ID No. 1 or a functional variant thereof.  
     
     
         7 . A test system according to  claim 6 , characterized in that the cNPK substrate is a peptide, polypeptide or peptoid comprising the pentamers containing amino acids 155-159 and/or 237-241 and/or 276-280 from SEQ ID No. 1 or the decamers containing amino acids 152-161 and/or 234-243 and/or 273-282 from SEQ ID No. 1.  
     
     
         8 . A test system according to  claim 1 , characterized in that the cNPK is preferably a cAMP kinase and/or cGMP kinase, or a functional variant thereof.  
     
     
         9 . A test system according to  claim 8 , characterized in that the cNPKs are present in purified form, in the form of blood extracts, cell extracts or tissue extracts, or in the form of permeabilized and/or intact cells.  
     
     
         10 . A test system according to any of  claims 1  to  9 , characterized in that the detection system for quantifying the phosphorylation of the cNPK substrate or the dephosphorylation of the VASP phosphatase substrate contains at least one antibody which is suitable for the phosphorylated product or for the dephosphorylated product.  
     
     
         11 . A test system according to  claim 10 , characterized in that the antibody is formed by the hybridoma cell line DSM ACC2330, and is, in particular, the monoclonal antibody 16C2 or a functional variant thereof.  
     
     
         12 . A test system according to  claim 10  or  11 , characterized in that the detection system comprises two antibodies, with the first antibody being unlabeled and the second antibody being labeled.  
     
     
         13 . A test system according to  claim 10  or  11 , characterized in that the detection system comprises at least one labeled antibody and also a suitable system for detecting the label.  
     
     
         14 . A test system according to  claim 10  or  11 , characterized in that the antibody contains a fluorophor.  
     
     
         15 . A test system according to any of  claims 1  to  14 , characterized in that the detection system is based on fluorescence resonance energy transfer (FRET), with use being made of at least one labeled, preferably one fluorophor-labeled, first antibody or second antibody and an acceptor fluorophor or donor fluorophor which corresponds thereto, preferably on the cNPK substrate.  
     
     
         16 . A test system according to  claim 15 , characterized in that the detection system is based on a time-resolved FRET system using lanthanide chelates, preferably europium chelates.  
     
     
         17 . A test system according to any of  claims 1  to  16 , characterized in that buffer solutions, stabilizers and/or energy equivalents are employed as further supplementary agents.  
     
     
         18 . A method for preparing a test system according to  claim 1 , characterized in that at least one compound to be investigated and at least one composition containing cNPK and ATP, and at least one detection system which is suitable for quantitatively determining the quantity of the phosphorylated cNPK substrate are combined.  
     
     
         19 . A method for preparing a test system according to  claim 2 , characterized in that at least one compound to be investigated, at least one composition containing VASP phosphatase and at least one detection system which is suitable for quantitatively determining the quantity of the dephosphorylated VASP phosphatase substrate are combined.  
     
     
         20 . An HTS-suitable method for finding one or more active compounds which modulate the activity of the cNPKs, comprising the steps of 
 i) bringing the compound to be investigated, or a multiplicity of compounds to be investigated, into contact with cNPK, ATP and a suitable cNPK substrate, and    j) quantifying the phosphorylated cNPK substrate using a suitable detection system.    
     
     
         21 . An HTS-suitable method for finding one or more active compounds which modulate the activity of the VASP phosphatase, comprising the steps of: 
 k) bringing the compound to be investigated, or a multiplicity of compounds to be investigated, into contact with VASP phosphatase and a suitable VASP phosphatase substrate, and    l) quantifying the dephosphorylated VASP phosphatase substrate using a suitable detection system.    
     
     
         22 . A method according to any of  claims 20  to  21 , characterized in that the compound to be investigated is selected from a naturally occurring, naturally occurring and chemically modified and/or synthetic compound.  
     
     
         23 . A method according to  claim 22 , characterized in that the compound to be investigated is used in the form of a combinatorial substance library.  
     
     
         24 . A method for diagnosing by directly, and in a gel electrophoresis-independent manner, determining the cNPK activity in samples, comprising the steps of: 
 m) incubating a sample in the presence of at least one composition containing a suitable cNPKS and ATP,    n) adding at least one detection system which is suitable for quantifying the phosphorylation of the cNPKS to the composition; and    o) determining the activity of the cNPK, in particular in blood extracts, cell extracts or tissue extracts or in samples which are in the form of permeabilized and/or intact cells.    
     
     
         25 . A method for diagnosing by directly, and in a gel electrophoresis-independent manner, determining the VASP phosphatase activity in samples, comprising the steps of: 
 p) incubating a sample in the presence of at least one composition containing a suitable VASP phosphatase substrate,    q) adding at least one detection system which is suitable for quantifying the dephosphorylation of the VASP phosphatase substrate to the composition; and    r) determining the activity of the VASP phosphatase, in particular in blood extracts, cell extracts or tissue extracts or in samples which are in the form of permeabilized and/or intact cells.    
     
     
         26 . A method according to  claim 24  or  25 , characterized in that the method is the diagnosis of a disease which is selected from the spectrum of angiocardiopathies and diseases which are associated with vascular damage, in particular hypertension, thrombosis and the endothelial dysfunction syndrome in arteriosclerosis, diabetes and vasculites, or diseases of hematopoietic cells, such as acute leukemia, myeloproliferative diseases or myelodysplasias.